The present invention relates to DNA constructs and plants incorporating them. In particular it relates to promoter sequences for the expression of genes which confer herbicide resistance on plants.
Recent advances in plant biotechnology have resulted in the generation of transgenic plants resistant to herbicide application. Herbicide tolerance has been achieved using a range of different transgenic strategies. One well documented example is the use the bacterial xenobiotic detoxifying gene phosphinothricin acetyl transferase (PAT) from Streptomyces hydroscopicus. Mutated genes of plant origin, for example the altered target site gene encoding acetolactate synthase (ALS) from Arabidopsis, have been successfully utilised to generate transgenic plants resistant to herbicide application. The PAT and ALS genes have been expressed under the control of strong constitutive promoter.
We propose a system where genes conferring herbicide tolerance would be expressed in an inducible manner dependent upon application of a specific activating chemical. This approach has a number of benefits for the farmer, including the following:
1. Inducible control of herbicide tolerance would alleviate any risk of yield penalties associated with high levels of constitutive expression of herbicide resistance genes. This may be a particular problem as early stages of growth where high levels of transgene product may directly interfere with normal development. Alternatively high levels of expression of herbicide resistance genes may cause a metabolic drain for plant resources.
2. The expression of herbicide resistance genes in an inducible manner allows the herbicide in question to be used to control volunteers if the activating chemical is omitted during treatment.
3. The use of an inducible promoter to drive herbicide resistance genes will reduce the risk of resistant weed species becoming a major problem. If resistance genes were passed onto weed species from related crops, control could still be achieved with the herbicide in the absence of inducing chemical. This would particularly be relevant if the tolerance gene confirmed resistance to a total vegetative control herbicide which would be used (with no inducing chemical) prior to sowing the crop and potentially after the crop has been harvested. For example, it can be envisaged that herbicide resistance in cereals, such as wheat, might outcross into the weed wild oats or that herbicide resistance in oil seed rape or canola could be transferred to wild brassicas thus conferring herbicide resistance to these already troublesome weeds. A further example is that the inducible expression of herbicide resistance in sugar beet will reduce the risk of wild sugar beet becoming a problem.
Several gene regulation systems (gene switches) are known and may be used for conferring inducible herbicide resistance on plants. Many such gene switches are described in the review by Gatz (Current Opinion in Biotechnology (1996) 7, 168-172) and include systems such as the tetracycline repressor gene switch, the Lac repressor system, copper inducible systems such as that based on ACE 1, salicylic acid inducible promoters including the PR-1a system and systems based on sterioid hormones such as the glucocorticoid, progesterone and oestrogen receptor systems. Modifications of the glucocorticoid receptor systems which include the GAL 4 binding domain from yeast and the VP 16 activator are described by Aoyama et al, The Plant Cell, (1995) 7, 1773-1785 and it is envisaged that similar systems may based on, for example insect steroid hormones rather than on mammalian steriod hormones. Indeed, a system based on the ecdysone receptor of Heliothis virescens has recently been described. Benzene sulphonamide gene switching systems are also known (Hershey et al, Plant Mol. Biol., 17, 679-690 (1991) as are systems based on the alcR protein from Aspergillus nidulans and glutathione S-transferase promoters.
Several genes which confer herbicide resistance are also known, for example, one herbicide to which resistance genes have been described and which is extremely widely used is N-phosphonomethyl-glycine (glyphosate) and its agriculturally acceptable salts including the isopropylamine, trimethylsulphonium, sodium, potassium and ammonium salts.
In a first aspect of the present invention there is provided a chemically inducible plant gene expression cassette comprising an inducible promoter operatively linked to a target gene which confers resistance to a herbicide.
Any herbicide resistance gene may be used but genes which confer resistance to N-phosphonomethyl-glycine or salts or derivatives thereof are especially preferred.
Several inducible promoters may be used to confer the inducible resistance and these include any of those listed above.
However, a particularly useful gene switch for use in this area is based on the alcR regulatory protein from Aspergilluis nidulans which activates genes expression from the alcA promoter in the presence of certain alcohols and ketones. This system is described in our International Patent Publication No. WO93/21334 which is incorporated herein by reference.
The alcA/alcR gene activation system from the fungus Aspergillus nidulans is also well characterised. The ethanol utilisation pathway in A. nidulans is responsible for the degradation of alcohols and aldehydes. Three genes have been shown to be involved in the ethanol utilisation pathway. Genes alcA and alcR have been shown to lie close together on linkage group VII and aldA maps to linkage group VIII (Pateman J H et al, 1984, Proc. Soc. Lond, B217:243-264; Sealy-Lewis E M and Lockington R A, 1984, Curr. Genet, 8:253-259). Gene alcA encodes ADHI in A. nidulans and aldA encodes aldDH, the second enzyme responsible for ethanol utilisation. The expression of both alcA and aldA are induced by ethanol and a number of other inducers (Creaser E H et al, 1984, Biochemical J. 255:449-454) via the transcription activator alcR. The alcR gene and a co-inducer are responsible for the expression of alcA and aldA since a number of mutations and deletions in alcR result in the pleiotropic loss of ADHI and aldDH (Felenbok B et al, 1988, Gene, 73:385-396; Pateman et al, 1984; Sealy-Lewis and Lockington, 1984). The ALCR protein activates expression from alcA by binding to three specific sites in the alcA promoter (Kulmberg P et al, 1992, J. Biol. Chem, 267:21146-21153).
The alcR gene was cloned (Lockington R A et al, 1985, Gene, 33:137-149) and sequenced (Felenbok et al, 1988). The expression of the alcR gene is inducible, autoregulated and subject to glucose repression mediated by the CREA repressor (Bailey C and Arst H N, 1975, Eur. J. Biochem, 51:573-577; Lockington R A et al, 1987, Mol. Microbiology, 1:275-281; Dowzer C E A and Kelly J M, 1989, Curr. Genet, 15:457-459; Dowzer C E A and Kelly J M, 1991, Mol. Cell. Biol, 11:5701-5709). The ALCR regulatory protein contains 6 cysteines near its N terminus coordinated in a zinc binuclear cluster (Kulmberg P et al, 1991, FEBS Letts, 280:11-16). This cluster is related to highly conserved DNA binding domains found in transcription factors of other ascomycetes. Transcription factors GAL4 and LAC9 have been shown to have binuclear complexes which have a cloverleaf type structure containing two Zn(II) atoms (Pan T and Coleman J E, 1990, Biochemistry, 29:3023-3029; Halvorsen Y D C et al, 1990, J. Biol. Chem, 265:13283-13289). The structure of ALCR is similar to this type except for the presence of an asymmetrical loop of 16 residues between Cys-3 and Cys-4. ALCR positively activates expression of itself by binding to two specific sites in its promoter region (Kulmberg P et al, 1992, Molec. Cell. Biol, 12:1932-1939).
The regulation of the three genes, alcR, alcA and aldA, involved in the ethanol utilisation pathway is at the level of transcription (Lockington et al, 1987; Gwynne D et al, 1987, Gene, 51:205-216; Pickett et al, 1987, Gene, 51:217-226).
There are two other alcohol dehydrogenases present in A. nidulans. ADHII is present in mycelia grown in non-induced media and is repressible by the presence of ethanol. ADHII is encoded by alcB and is also under the control of alcR (Sealy-Lewis and Lockington, 1984). A third alcohol dehydrogenase has also been cloned by complementation with a adh-strain of S. cerevisiae. This gene alcC, maps to linkage group VII but is unlinked to alcA and alcR. The gene, alcC, encodes ADHIII and utilises ethanol extremely weakly (McKnight G L et al, 1985, EMBO J, 4:2094-2099). ADHIII has been shown to be involved in the survival of A. nidulans during periods of anaerobic stress. The expression of alcC is not repressed by the presence of glucose, suggesting that it may not be under the control of alcR (Roland L J and Stromer J N, 1986, Mol. Cell. Biol, 6:3368-3372).
In summary, A. nidulans expresses the enzyme alcohol dehydrogenase I (ADH1) encoded by the gene alcA only when it is grown in the presence of various alcohols and ketones. The induction is relayed through a regulator protein encoded by the alcR gene and constitutively expressed. In the presence of inducer (alcohol or ketone), the regulator protein activates the expression of the alcA gene. The regulator protein also stimulates expression of itself in the presence of inducer. This means that high levels of the ADH1 enzyme are produced under inducing conditions (i.e. when alcohol or ketone are present). Conversely, the alcA gene and its product, ADH1, are not expressed in the absence of inducer. Expression of alcA and production of the enzyme is also repressed in the presence of glucose.
Thus the alcA gene promoter is an inducible promoter, activated by the alcR regulator protein in the presence of inducer (i.e. by the protein/alcohol or protein/ketone combination). The alcR and alcA genes (including the respective promoters) have been cloned and sequenced (Lockington R A et al, 1985, Gene, 33:137-149; Felenbok B et al, 1988, Gene, 73:385-396; Gwynne et al, 1987, Gene, 51:205-216).
Alcohol dehydrogenase (adh) genes have been investigated in certain plant species. In maize and other cereals they are switched on by anaerobic conditions. The promoter region of adh genes from maize contains a 300 bp regulatory element necessary for expression under anaerobic conditions. However, no equivalent to the alcR regulator protein has been found in any plant. Hence the alcR/alcA type of gene regulator system is not known in plants. Constitutive expression of alcR in plant cells does not result in the activation of endogenous adh activity.
According to a second aspect of the invention, there is provided a chemically-inducible plant gene expression cassette comprising a first promoter operatively linked to an alcR regulator sequence which encodes an alcR regulator protein, and an inducible promoter operatively linked to a target gene which confers herbicide resistance, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer a whereby application of the inducer causes expression of the target gene.
The inducible promoter is preferably derived from the alcA gene promoter but may, alternatively be derived from alcR, aldA or other alcR-induced genes.
We have found that the alcA/alcR switch is particularly suited to drive herbicide tolerance genes for at least the following reasons.
1. The alcA/alcR switch has been developed to drive high levels of gene expression. In addition, the regulatory protein alcR is preferably driven from a strong constitutive promoter such as polyubiquitin. High levels of induced transgene expression, comparable to that from a strong constitutive promoter, such as 35 CaMV, can be achieved.
2. If a gene switch is to be used in a situation where the activating chemical is applied simultaneously with the herbicide, a rapid elevation in the levels of herbicide resistance gene is required. FIG. 1 reveals a time course of marker gene expression (CAT) following application of inducing chemical. This study shows a rapid increase (2 hours) of CAT expression following foliar application of inducing chemical. The immediate early kinetics of induction are brought about be expressing the regulatory protein in constitutive manner, therefore no time lag is encountered while synthesis of transcription factors takes place. In addition we have chosen a simple two component system which does not rely on a complex signal transduction system.
3. We have tested the specificity of alcA/alcR system with a range of solvents used in agronomic practice. A hydroponic seedling system revealed that ethanol, butan-2-ol and cyclohexanone all gave high levels of induced reporter gene expression (FIG. 2). In contrast when the alcohols and ketones listed in Table 1 in which are used in agronomic practice were applied as a foliar spray only ethanol gave high levels of induced reporter gene activity (FIG. 3).
This is of significance since illegitimate induction of transgenes will not be encountered by chance exposure to formulation solvents. Ethanol is not a common component of agrochemical formulations and therefore with appropriate spray management can be considered as a specific inducer of the alc A/R gene switch in a field situation.
4. A range of biotic and abiotic stresses for example pathogen infection, heat, cold, drought, wounding, flooding have all failed to induce the alcA/alcR switch. In addition a range of non-solvent chemical treatments for example salicylic acid, ethylene, absisic acid, auxin, gibberelic acid, various agrochemicals, all failed to induce the alcA/alcR system.
The first promoter may be constitutive or tissue-specific, developmentally-programmed or even inducible. The regulator sequence, the alcR gene, is obtainable from Aspergilluis nidulans, and encodes the alcR regulator protein.
The inducible promoter is preferably the alcA gene promoter obtainable from Aspergilluis nidulans or a xe2x80x9cchimericxe2x80x9d promoter derived from the regulatory sequences of the alcA promoter and the core promoter region from a gene promoter which operates in plant cells (including any plant gene promoter). The alcA promoter or a related xe2x80x9cchimericxe2x80x9d promoter is activated by the alcR regulator protein when an alcohol or ketone inducer is applied.
The inducible promoter may also be derived from the aldA gene promoter, the alcB gene promoter or the alcC gene promoter obtainable from Aspergillus nidulans. 
The inducer may be any effective chemical (such as an alcohol or ketone). Suitable chemicals for use with an alcA/alcR-derived cassette include those listed by Creaser et al (1984, Biochem J, 225, 449-454) such as butan-2-one (ethyl methyl ketone), cylcohexanone, acetone, butan-2-ol, 3-oxobutyric acid, propan-2-ol, ethanol.
The gene expression cassette is responsive to an applied exogenous chemical inducer enabling external activation of expression of the target gene regulated by the cassette. The expression cassette is highly regulated and suitable for general use in plants.
The two parts of the expression cassette may be on the same construct or on separate constructs. The first part comprises the regulator cDNA or gene sequence subcloned into an expression vector with a plant-operative promoter driving its expression. The second part comprises at least part of an inducible promoter which controls expression of a downstream target gene. In the presence of a suitable inducer, the regulator protein produced by the first part of the cassette will activate the expression of the target gene by stimulating the inducible promoter in the second part of the cassette.
In practice the construct or constructs comprising the expression cassette of the invention will be inserted into a plant by transformation. Expression of target genes in the construct, being under control of the chemically switchable promoter of the invention, may then be activated by the application of a chemical inducer to the plant.
Any transformation method suitable for the target plant or plant cells may be employed, including infection by Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, microprojectile transformation and pollen tube transformation. The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way.
Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.
The invention further provides a plant cell containing a gene expression cassette according to the invention. The gene expression cassette may be stably incorporated in the plant""s genome by transformation. The invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom.
The invention further provides a method for controlling plant gene expression comprising transforming a plant cell with a chemically-inducible plant gene expression cassette which has a first promoter operatively linked to an alcR regulator sequence which encodes an alcA regulator protein, and an inducible promoter operatively linked to a target gene which confers herbicide resistance, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.
This strategy of inducible expression of herbicide resistance can be achieved with a pre-spray of chemical activator or in the case of slow acting herbicides, for example N-phosphonomethyl-glycine (commonly known as glyphosate), the chemical inducer can be added as a tank mix simultaneously with the herbicide.
This strategy can be adopted for any resistance conferring gene/corresponding herbicide combination. For example, the alcA/alcR gene switch can be used with:
1. Maize glutathione S-transferase (GST-27) gene (see our International Patent Publication No WO90/08826), which confers resistance to chloroacetanilide herbicides such as acetochlor, metolachlor and alachlor.
2. Phosphinotricin acetyl transferase (PAT), which confers resistance to the herbicide commonly known as glufosinate.
3. Acetolactate synthase gene mutants from maize (see our international Patent Publication No WO90/14000) and other genes, which confer resistance to sulphonyl urea and imadazlonones.
4. Genes which confer resistance to glyphosate. Such genes include the glyphosate oxidoreductase gene (GOX) (see International Patent Publication No. WO92/00377 in the name of Monsanto Company); genes which encode for 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSPS), including Class I and Class II EPSPS, genes which encode for mutant EPSPS, and genes which encode for EPSPS fusion peptides such as that comprised of a chloroplast transit peptide and EPSPS (see for example EP 218 571, EP 293 358, WO91/04323, WO92/04449 and WO92/06201 in the name of Monsanto Company); and genes which are involved in the expression of CPLyase.